In brain, pH regulation is important because many ion channels, neurotransmitter-uptake systems, and cellular processes are sensitive to changes in pH. To regulate both intracellular pH (pHi) and extracellular Ph (pHo), brain cells such as neurons and glia utilize plasma-membrane transporters to shuttle hydrogen and bases such as bicarbonate across their membranes. Bicarbonate-coupled transporters such as Na/Bicarbonate Cotransporters (NBCs) are particularly potent regulators of brain pH, and electrogenic ones occupy an unusual niche in contributing to activity-evoked changes in pH. Our understanding of this family of proteins has expanded following the molecular identification of many of these proteins. The long-term objective of this proposal is to elucidate the physiological significance of multiple electrogenic NBCs in brain. Antibodies to specific isoforms will be used to examine the expression profiles of the proteins throughout the rat brain (Aim 1). Subsequently, the contribution of each NBC to the pH physiology of the rat hippocampus will be explored. The identification and biophysical characterization of NBC activity in each hippocampal cell type will be revealed and compared using fluorescence imaging of pHi and patch-clamp techniques (Aim 2). Functional properties will therefore be assigned to the molecules identified in Aim 1. Finally, to elucidate the structural features that underlie the functional properties of the NBCe1 proteins studied in Aim 2, structure-function relationships of NBCe1 will be examined (Aim 3). Utilizing available information on related anion exchangers (AEs), specific regions and residues responsible for ion binding and translocation, as well as pH and voltage sensitivities will be determined. Chimeric NBC-AEs and truncated/mutant NBCs will be expressed and functionally characterized in oocytes using microelectrodes and the macropatch technique, as well as in transfected mammalian cells using fluorescence imaging. Studies will involve examining fundamental bicarbonate transport, as well as the more detailed transport properties of ion, pH, and voltage dependencies and activation kinetics. Results will provide information pertinent to other members of the superfamily. Understanding the expression, function, and structure of electrogenic NBCs will help clarify the influence of these proteins on neuronal activity and synaptic transmission, as well as their involvement in acid-base disturbances such as epilepsy, ischemia, and hypoxia.